JoVE Journal > Genetics
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Genetics

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

Published: June 30, 2022
doi:
10.3791/63232
1College of Arts and Sciences,Northwest University

Summary

ACT1-CUP1测定是一种铜生长测定，可快速读取前体信使RNA（pre-mRNA）剪接以及突变剪接因子对剪接体功能的影响。本研究提供了一个协议，并强调了解决感兴趣的拼接问题的定制可能。

Abstract

剪接体或其底物中引入的突变极大地促进了我们对剪接体功能复杂性的理解。无论是疾病相关还是功能选择，许多这些突变都已使用模式生物酿 酒 酵母（酵母）的生长测定进行了研究。剪接特异性铜生长测定或ACT1-CUP1测定在表型水平上提供了对突变的全面分析。ACT1-CUP1测定利用报告基因在正确拼接时赋予铜公差。因此，在铜存在的情况下，酵母活力的变化与剪接中mRNA产生的变化相关。在典型的实验中，酵母剪接体受到不同的非共识剪接报告基因和感兴趣的剪接因子突变的挑战，以检测对剪接的任何协同或相反影响。这里给出了铜板制备、酵母细胞电镀和数据评估的完整描述。描述了一系列免费的实验，突出了ACT1-CUP1报告基因的多功能性。ACT1-CUP1测定是剪接工具箱中方便的工具，这要归功于直接读取突变效应和在该领域继续使用的比较可能性。

Introduction

剪接体是一种大型生物机器，可催化去除前体信使RNA（pre-mRNA）中的内含子和非编码区域1，2。在单独研究蛋白质或RNA时，表征近100种蛋白质和5种非编码RNA中的1种中的单点突变体的作用通常是模棱两可的。突变组件功能的变化可以在完整的功能剪接体的背景下最好地在体内进行评估。

这里描述的铜生长测定是 酿酒 酵母或出芽酵母剪接效率的快速衡量标准。该测定由C.F. Lesser和C. Guthrie开发并于1993年发表，结合了与简单模式生物的易用性和细胞活力的直接读数3。活力与这些细胞中的剪接体识别和拼接报告转录本的能力相关。

这种铜生长测定通常称为ACT1-CUP1测定。ACT1-CUP1这个名字起源于两个基因融合在一起，创造了剪接效率的报告基因。ACT1是酵母的肌动蛋白基因，其高表达并具有有效的剪接内含子4，5。Cup1p是一种铜螯合剂，可将铜隔离在细胞中，以防止干扰正常的细胞功能6，7，8。ACT1-CUP1报告基因按顺序包含这些基因，使得只有当ACT1内含子发生前mRNA剪接时，CUP1才处于正确的阅读框中（图1）。所得融合蛋白含有肌动蛋白的前 21 个氨基酸和全长 Cup1p 蛋白，可提高酵母在富含铜的环境中的活力3.因此，报告基因拼接量的增加导致更高的Cup1p浓度和更高的铜电阻（图1）。与其他报告基因相比，CUP1即使在低水平下也会影响细胞活力，具有广泛的灵敏度范围，可用于直接选择剪接突变3，6，7。此外，CUP1对于标准酵母生长不是必需的，因此在该测定的设置过程中不会影响细胞稳态。作为缺失或温度生长测定的补充，ACT1-CUP1提供了有关在其他最佳酵母生长条件下对剪接的影响的信息。

剪接体通过三个内含子序列识别其底物，即5’剪接位点（5’SS）、分支位点（BS）和3’剪接位点（3’SS）。已经产生了许多ACT1-CUP1报告基因，其中包含这些位点的非共识序列。 最常见的 ACT1-CUP1报告基因的选择如图1和 表1所示。由于剪接体在剪接周期的不同点与每个剪接位点唯一地相互作用，因此可以根据使用非共识报告基因的不同步骤测试剪接体的稳健性。非共识报告基因以内含子内的突变位置及其突变的碱基命名。例如，A3c是一个在5’SS处发生突变的报告基因，特别是从共识腺苷到胞嘧啶的位置3。该报告将与影响5’SS选择和使用的剪接体突变发生强烈相互作用。在他们最初的研究中，Lesser和Guthrie确定了哪些5’SS突变抑制剪接3。同年晚些时候，Burgess和Guthrie在ATPase Prp16p9突变的抑制筛选中发表了所有三个剪接位点的非共识报告。将共识与非共识报告基因进行比较，ACT1-CUP1测定是了解酵母剪接体的稳健性和选择性以及推断其他真核生物剪接体功能的重要关键。

由于非共识ACT1-CUP1报告基因使剪接体对进一步扰动敏感，因此可以通过报告基因来表征单个剪接因子突变的影响，其积极影响或负面影响。这已被应用于以各种方式拼接研究问题。首先，ACT1-CUP1测定可以并且已经被用作剪接因子突变的遗传筛选。例如，最大的剪接蛋白Prp8p作为剪接体RNA核心催化剪接反应的平台。这部分是通过Prp8p突变体如何改善或减少不同ACT1-CUP1报告基因10，11，12，13，14，15，16，17的剪接来推断的。剪接体的其他蛋白质成分也已使用ACT1-CUP1进行了研究，包括Hsh155p，Cwc2p，Cef1p和Ecm2p18，19，20，21，22，23，24，25。Prp16p和其他四种参与剪接体转化的ATP酶的能量阈值也已通过该测定9，26，27，28，29，30进行了研究。利用ACT1-CUP1对小核RNA（snRNA）进行了广泛的研究，以鉴定它们协调的前mRNA序列以及snRNA在剪接过程中经历的二级结构的变化3，31，32，33，34，35，36，37。

ACT1-CUP1测定需要酵母菌株，其中CUP1基因的所有拷贝都被敲除。由于CUP1可以具有高拷贝数6，38，因此制备完整的敲除菌株可能需要多轮或广泛的筛选。因此，cup1Δ酵母菌株经常在实验室之间共享，记者也是如此。

如果从质粒拷贝评估剪接因子中的突变，则应敲除该因子的野生型基因。此外，酵母背景应允许选择至少两个质粒，一个包含ACT1-CUP1报告基因，历史上在亮氨酸营养选择质粒上，另一个包含将要研究的剪接机制中的突变或扰动（图2）。通常，在一次测定中，多个酵母菌株，每个菌株携带查询剪接扰动（QSP）和不同的报告基因，将测试查询对剪接的影响。

ACT1-CUP1测定中的自变量允许研究人员评估QSP的严重程度。这些自变量是铜的浓度和多个非共识拼接报告基因的选择。首先，由于酵母菌株生长在含有一系列铜浓度的平板上（图2），因此设置测定包括选择所用浓度的梯度。研究可以利用过程铜浓度梯度来获得活性的初始读数，然后以更精细的梯度重复测定以确定细微的活性差异。第二个变量是可以测试的ACT1-CUP1报告基因的广泛范围（图 1和 表1）。如果QSP在非共识报告基因与野生型存在的情况下对酵母活力的影响不同，则可以得出结论，QSP影响剪接步骤或剪接体中在识别或处理内含子该区域期间重要的区域。

酵母工具箱非常广泛，ACT1-CUP1测定是剪接研究的一个组成部分。ACT1-CUP1测定通常与对QSP影响的更深入的遗传，结构和/或生化分析一起进行。由于这些更详细的研究通常具有更长的程序和/或更高的价格标签，因此常见的方法是首先筛选具有ACT1-CUP1的有趣突变体。

这里提供的是ACT1-CUP1测定方案，包括铜板制备。该测定为研究人员提供了QSP对剪接影响的初步答案，以及哪些内含子区域受扰动的影响最大。

Protocol

1. 酵母菌株构建 生成或获得酿 酒酵母 菌株，其背景包括 leu2 和 cup1Δ。要生成此背景，请使用采用乙酸锂和单链DNA39的成熟酵母方法。注意：单倍体酵母菌株可能包含一个，两个或多个 CUP16，38拷贝。在设计敲除引物以侧翼 CUP1 基因位置时，请参阅所选酵母菌株的基因组信息。</…

Representative Results

生长测定，如ACT1-CUP1，需要对多个菌落进行目视比较评估。在这里，将每种菌株在一夜之间生长至饱和，稀释至OD600 为0.5，并镀在含有0 mM至1.1 mM CuSO4 铜浓度范围的20个板上（图3）。该范围小于协议中列出的范围，因为它允许全面评估下面使用和描述的QSP和ACT1-CUP1报告的影响。对板进行成像和评分（图 3和 补充表3）。 <p…

Discussion

ACT1-CUP1是一种生长测定，必须注意确保观察到的生长差异只能归因于剪接缺陷。所有菌株在铺板前应以类似的方式处理，包括具有相似的长度和类型的生长和储存条件。如果使用温度敏感菌株，ACT1-CUP1测定应仅在这些菌株生长与野生型相当的条件下进行。相关地，对于QSP成分，建议具有相同的酵母背景和QSP基因的表达水平，以免影响结果的解释。在考虑要使用的QSP和报告器数量时，不建议使用针?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

感谢Aaron Hoskins和威斯康星大学麦迪逊分校的Hoskins实验室成员在生成图3-5时使用酵母菌株和设备。感谢Harpreet Kaur和傅兴阳对手稿的深刻评论。感谢西北大学在撰写、编辑和拍摄本文期间给予的支持。感谢Isabelle Marasigan在拍摄这种方法时提供的帮助。

Materials

1.5 mL sterile microcentrifuge tubes Fisher Scientific 05-408-129 Or comparable item from a different manufacturer.
2 mL sterile microcentrifuge tubes Fisher Scientific 05-408-138 Or comparable item from a different manufacturer.
50 mL sterile centrifuge tubes Fisher Scientific 07-201-332 Or comparable item from a different manufacturer.
96-well round bottom microplate Fisher Scientific 07-200-760 Or comparable item from a different manufacturer.
190 proof ethanol Fisher Scientific 22-032-600 Or comparable item from a different manufacturer.
500 mL Filter System (0.22 µm) CellTreat Scientific Products 229707 Or comparable item from a different manufacturer.
Agar Fisher Scientific BP1423-500 Any molecular grade agar will work.
Autoclave Tuttnauer 3870EA Or comparable item from a different manufacturer.
Bunsen burner Humboldt PN6200.1 Or comparable item from a different manufacturer.
Cell Density Meter VWR 490005-906 Or other spectral device that can measure absorbance at 595 nm.
Copper sulfate Pentahydrate Fisher Scientific LC134051 Or comparable item from a different manufacturer.
Digital imaging system Cytiva 29399481 ImageQuant 4000 (used for Figure 3),  Amersham ImageQuant 800, or comparable item from a different manufacturer.
Dropout mix (-Leu) USBiological Life Sciences D9525 Use the appropriate drop out mix for your experiment. It is possible you will be using a yeast nutrient marker for your query perturbation also. In that case, the drop out mix should be for that marker and Leu
D-Glucose Fisher Scientific AAA1682836 Or comparable item from a different manufacturer.
Gel band quantifying software Cytiva 29-0006-05 ImageQuant TL v8.1 (used for figure 5A) or comparable item from a different manufacturer.
Hand held camera Nikon D3500 Or comparable item from a different manufacturer.
Near infra-red gel imaging device Cytiva 29238583 Amersham Typhoon NIR (used for Figure 5a) or comparable item from a different manufacturer.
Laboratory grade clamp Fisher Scientific 05-769-7Q Or comparable item from a different manufacturer.
Laboratory grade stand and clamp Fisher Scientific 12-000-101 Or comparable item from a different manufacturer.
Magnetic stir bars Fisher Scientific 14-513-51 Or comparable item from a different manufacturer.
Pin replicator VP Scientific VP 407AH
Semi-micro disposable cuvettes VWR 97000-590 Or comparable item from a different manufacturer.
Shaker JEIO Tech IST-3075 Or comparable item from a different manufacturer.
Spectrophotometer Biowave 80-3000-45 Or any spectophotometer that can measure the absorbance at 600 nm.
Square plates VWR 102091-156 Circular plates may also be used though are more challenging if using a pin replicator.
Stir plate Fisher Scientific 11-520-16S Or comparable item from a different manufacturer.
Yeast nitrogen base USBiological Life Sciences Y2025 Or comparable item from a different manufacturer.
DOWNLOAD MATERIALS LIST

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ACT1-CUP1 AssayPre-messenger RNA SplicingSplicing Factor MutationGrowth PhenotypeLeucine Dropout Growth MediaCopper SulfateOptical DensitySpectrophotometerDilutionBunsen BurnerReplicator
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van der Feltz, C. ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast. J. Vis. Exp. (184), e63232, doi:10.3791/63232 (2022).
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